基于MLPA和DHPLC技术平台的家族性腺瘤息肉病家系致病性APC基因胚系突变筛查及突变分子机制分析

发布时间：2018-04-30 19:20

本文选题：家族性腺瘤息肉病 + APC　；参考：《南京医科大学》2015年硕士论文

【摘要】：目的:通过基因诊断技术寻找和分析家族性腺瘤息肉病(familial adenomatous polyposis,FAP)家系结肠腺瘤息肉病(adenomatous polyposis coli,APC)基因致病性胚系突变及其特点,同时检测基于多重连接依赖性探针扩增(multiplex ligationdependent probe amplification,MLPA)和变性高效液相色谱技术(denaturing High Performance Liquid Chromatography,DHPLC)技术平台的基因诊断效力。方法:提取2005年至2013就诊于我院的14个FAP家系成员外周血DNA,联合应用MLPA、PCR-DHPLC技术及直接测序等方法对APC基因胚系突变进行检测,寻找各家系APC基因致病性胚系突变。随后分析突变位点周围碱基序列,探索APC基因胚系突变特点。结果:14个家系中有6个家系筛查出了APC基因胚系突变,分别是c.5432CT(p.Ser1811Leu)、两个c.3926_3930del AAAAG(p.Glu1309Aspfs X4)、c.3921_3924del AAAA(p.Ile1307Metfs X13)、c.3184_3187del CAAA(p.Gln1061Aspfs X59)和c.4127_4126del AT(p.Tyr1376Lysfs X9),所有的缺失突变都导致了截断蛋白的产生。14个家系中均未发现有大片段缺失与重复。分析缺失突变位点周围碱基序列发现,c.3926_3930del AAAAG与c.3921_3924del AAAA位于AAAAG短串联重复序列中,c.3184_3187del CAAA位于间断的短串联重复序列中,而c.4127_4128del AT则位于5'-CCTGAACA-3',3'-ACAAGTCC-5回文序列(反向重复序列),并且大部分的突变位于密码子1309周围。结论:包含短串联重复序列及回文序列的区域为APC基因致病性胚系突变高发区域,以小片段缺失为主,尤其是密码子1309位点的5碱基缺失最为常见。基于MLPA和DHPLC技术平台的基因诊断技术可快速、高效地进行基因突变筛查。
[Abstract]:Objective: to identify and analyze the mutation and characteristics of pathogenic embryoid line of adenomatous polyposis in a family of familial adenomatous polyposis (adenomatous polyposis) by means of gene diagnosis technique. At the same time, the gene diagnostic efficacy of multiplex ligationdependent probe amplification (MLPA) and denaturing High Performance Liquid chromatography (DHPLC) platform based on multiplex ligationdependent probe amplification and denaturing high performance liquid chromatography (denaturing High Performance Liquid) were also detected. Methods: peripheral blood DNA was extracted from 14 FAP family members from 2005 to 2013 in our hospital. APC gene embryogenic mutation was detected by MLPA-PCR-DHPLC and direct sequencing. The pathogenicity of APC gene was found in all families. Then the base sequence around the mutation site was analyzed to explore the characteristics of APC gene blastocyst mutation. Results: in 6 of 14 families, APC gene embryonal mutations were screened. The results were as follows: C. 5432CTp.Ser1811 Leuer, two c.3926_3930del AAAAG(p.Glu1309Aspfs X4s, c. 3921 3924del AAAA(p.Ile1307Metfs X13 / c. 31844 del CAAA(p.Gln1061Aspfs X59) and c.4127_4126del AT(p.Tyr1376Lysfs X9, all deletion mutations resulted in the production of truncated proteins. No large deletion or duplication was found in 14 families. By analyzing the base sequence around the deletion mutation site, we found that AAAAG and c.3921_3924del AAAA are located in the AAAAG short tandem repeat sequence, and the c. 3184 CAAA is located in the discontinuous short tandem repeat sequence. The c.4127_4128del AT was located in the palindrome sequence (reverse repeat sequence) of 5CT-CCGAACA-3ACAAGTCC-5, and most of the mutations were located around codon 1309. Conclusion: the region containing short tandem repeats and palindromes is a high incidence region of APC gene pathogenicity embryo line mutation. Small fragment deletion is the most common, especially the 5-base deletion at codon 1309. Gene diagnosis technology based on MLPA and DHPLC technology platform can quickly and efficiently screen gene mutation.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735

【参考文献】